The present invention relates to the generation, by recombinant DNA methods, of novel immunoglobulins specific for the Lewis Y antigen, using, as a starting point, a murine monoclonal antibody against the same antigen.
Transformation of a normal cell to a malignant cell is often accompanied by a change in the expression of cell surface antigens. These different phenotypes can be detected using monoclonal antibodies specific for such antigens. In this way, different cancer cells can be detected and characterised (Lloyd, K. O. (1983) "Human Tumour Antigens: Detection and Characterisation with Monoclonal Antibodies" in R. B. Herberman, ed., Basic and Clinical Tumour Immunology, pp 159-214, Martinus Nijhoff, Boston). Lewis Y is a carbohydrate antigen with the structure Fuc.alpha.1.fwdarw.2Gal.beta.1.fwdarw.4[Fuc.alpha.1.fwdarw.3]GlcNAc.beta.1 .fwdarw.3R (Abe et al. (1983) J. Biol. Chem. 258 11793-11797). The Lewis Y antigen is expressed in normal tissues but the level of expression is higher in certain tumour types so that the antigen can be used as a marker for cells of some breast, colon, gastric, esophageal, pancreatic, duodenal, lung, bladder and renal carcinomas and gastric and islet cell neuroendocrine tumours. Its presence on some tumour cells is not accompanied by an increase in its serum levels, thus administered Lewis Y specific antibody is not significantly bound by soluble antigen.
Although a murine monoclonal antibody reactive against the Lewis Y antigen has potential in the imaging and treatment of certain tumours in man, this potential may be difficult to realise due to the xenogenic nature of the antibody. The problems associated with this are two-fold. First, administered xenogenic antibodies are likely to be immunogenic (Bruggemann et al (1989) J. Exp. Med. 170, 2153-2157). In this case, this would cause a human anti-mouse antibody (HAMA) response (Schroff, R. et al (1985) Cancer Res. 45, 879-885), resulting in rapid clearance of the antibody from the circulation. Second, depending upon the isotypes involved, a murine antibody may be less efficacious than a human counterpart in the stimulation of human complement or cell-mediated cytotoxicity.
To secure the advantage of a human antibody, whilst making use of the antigen-binding properties of an antibody raised in a different species, workers have made use of recombinant DNA techniques. EP120694 (Celltech) and EP125023 (Genentech) disclose the development of `chimaeric` antibodies which comprise the variable regions of an antibody from another species and the constant regions of a human antibody. Such chimaeric antibodies have the advantage since they retain the specificity of the murine antibody but can also stimulate human Fc dependent complement fixation and cell-mediated cytotoxicity. However, the murine variable regions can still elicit a HAMA response (Bruggemann, M. et al (1989) J.Exp.Med. 170, 2153-2157) thereby limiting the value of chimaeric antibodies as diagnostic and therapeutic agents.
British Patent Application Number GB2188638A (Winter) discloses the process of humanization in which only the antigen binding-loops are transferred to a human antibody template (for example Riechmann et al., Nature, 332, 323-327; Tempest et al., (1991) Bio/Technology 9, 266-271). These loops, known as complementary-determining regions (CDRs), are mounted on a scaffold--the frameworks regions. Together these make up the variable domains. Each binding site is formed, in the most part, from three heavy chain and three light chain CDRS, although framework residues can interact with antigen, either directly or indirectly, by altering the CDR conformation. `Reshaped` or `humanized` antibodies made by the process of CDR-grafting retain the human constant regions necessary for the stimulation of human Fc-dependant effector functions and have less murine content than chimaeric antibodies, consequently, humanized antibodies are less likely than chimaeric antibodies to evoke a HAMA response when administered to humans, their half-life in circulation should approach that of natural human antibodies, thereby enhancing their diagnostic and therapeutic value.
In practice, for the generation of efficacious humanized antibodies retaining the specificity of the original murine antibody, it is not usually sufficient simply to substitute CDRs. There is a requirement for the inclusion of a small number of critical murine antibody residues in the human variable region frameworks. The identity of these residues depends on the structure of both the original murine antibody and the acceptor human antibody.
The present invention provides novel humanized monoclonal antibodies specific for the Lewis Y antigen. This has been achieved by the generation of a murine monoclonal antibody reactive to the Lewis Y antigen, followed by utilisation of its variable domain structures in the design of the recombinant antibodies. Prior to the work of the inventors, it was not known that S193S or any other non-human antibody specific for the the Lewis Y antigen could be humanized so as to retain useful binding specificity.